Introduction: Biallelic mutations in LRRC33 cause a currently untreatable childhood-onset neurological disease that we term LRRC33-related encephalopathy (LRE). LRRC33 is expressed by intracranial macrophages, including highly specialised microglia of the brain parenchyma. LRE is a suspected microgliopathy—a type of brain disease driven directly by dysfunctional microglia—however fundamental questions regarding the cell-type specificity, timing, phenotypic diversity, and mechanisms of microglial dysfunction resulting from defective LRRC33 are unanswered.

Aims: This study aims to characterise brain macrophage phenotypes in human LRE brain tissue, and to determine the temporal, spatial, and mechanistic alterations of microglia in a preclinical model of the disease. Furthermore, we seek to investigate whether therapeutic strategies aimed at eliminating or replacing microglia can mitigate disease-associated pathology.

Methods: Using human post-mortem brain tissue, as well as Lrrc33 and Tgfb1 deficient mouse models, we characterised microglial phenotypes by histology (RNAscope/IHC; n = 3-12 per group), in vitro assays (n = 4-7 per group), and scRNA-seq (n = 2-4 mice per group). Therapeutic potential was assessed in mice through pharmacological microglial elimination (n = 7-11 per group) and bone marrow transplantation (n = 3-8 per group), followed by behavioural assays. Where appropriate, data were analysed using linear mixed-effects models or ANOVA with post-hoc corrections for multiple testing to account for fixed and random effects. Two-sided tests were used throughout. P (or adjusted P) < 0.05 was considered significant. All animal procedures were performed in a UK Home Office–licensed facility in accordance with the Animal (Scientific Procedures) Act 1986.

Results: We show in both humans and mice that microglia, but not other intracranial macrophages, fail to specialise in the absence of functional LRRC33, instead giving rise to abnormal microglia distinct from all described profiles in normal physiology or other diseases. In Lrrc33-/- mice, sustained elimination of microglia, or their replacement with Lrrc33-expressing transplanted donor myeloid cells, rescues disease, demonstrating that dysfunctional microglia are actively harmful and drive LRE, and revealing a clinically relevant therapeutic approach. Mechanistically, microglial-restricted deletion of transforming growth factor (TGF)-β1 induces developmental defects equivalent to those caused by LRRC33 deficiency, implicating LRE as a neurodevelopmental disorder rooted in failed microglia-intrinsic TGF-β1 signalling.

Conclusions: Our findings identify abnormal microglia as central drivers of LRE, establish it as a distinctive early-onset microgliopathy, and demonstrate that an intact LRRC33–TGF-β1 pathway within microglia is essential for normal human brain development.